Precise staging of Caenorhabditis elegans is essential for developmental studies in different environmental conditions. In favorable conditions, larvae develop continuously through four larval stages separated by molting periods. Distinguishing molting from intermolt larvae has been achieved using transgenes with molting reporters, therefore requiring strain constructions, or careful observation of individuals for pharyngeal pumping or behavioral quiescence. In unfavorable conditions, larvae can enter the stress-resistant and developmentally arrested dauer larva stage. Identifying dauer larvae has been based on their ability to withstand detergent selection, precluding identification of recovering animals or of mutants with defects in dauer morphogenesis. Here, we describe a simple method to distinguish molting larvae or dauer larvae from intermolt larvae that bypasses the limitations of current methods. Fluorescent latex beads are mixed with the bacterial food source and ingested by intermolt larvae and adults. Molting and dauer larvae do not feed, and therefore lack beads in their digestive tract. The presence of beads can be determined using a dissecting microscope at magnifications as low as 100 ×, or by using a wormsorter for high-throughput experiments. We find that continuously developing bead-lacking larvae display hallmarks of molting, including expression of the mlt-10::gfp molting marker and a lack of pharyngeal pumping. Furthermore, wild-type and mutant dauer larvae produced by any of three common methods are accurately identified by a lack of beads. Importantly, this method is effective in SDS-sensitive mutant backgrounds and can identify recovering dauer larvae, a stage for which there is no other method of positive selection.
Keywords: C. elegans; dauer; developmental stage; fluorescent beads; molting.
Copyright © 2016 Nika et al.